Metastasis is one of the hallmark behaviors of cancer and understanding the regulatory processes controlling it is key to improving outcomes for solid tumor patients. However, regulatory pathways affecting metastasis often affect other hallmark behaviors of cancer, i.e. proliferation, making it difficult to isolate mechanisms essential to metastasis. One disease in which metastasis is the primary cause of morbidity and mortality is osteosarcoma, most common primary bone cancer, with a peak incidence during adolescence. Osteosarcoma also has served excellent model system for studying common malignant behaviors. Recently we discovered a novel role for Notch signaling, especially the Notch target gene Hes1, specifically in promoting osteosarcoma metastasis. We showed Hes1 expression is essential for in vitro invasion and in vivo metastasis of osteosarcoma, while other hallmark behaviors of cancer were unaffected by Hes1 changes. This metastasis-promoting effect of Hes1 was not mediated by MMPs, VEGF, Ezrin or other recognized mediators of metastasis, but rather represents a novel regulatory mechanism important for controlling metastasis and invasion. Thus the focused role of Hes1 in promoting invasion and metastasis of osteosarcoma represents a unique and exciting opportunity to evaluate this new regulatory mechanism in a system where the effect of the gene is limited to metastasis. Given that Notch pathway expression confers a worse prognosis on many solid tumors, our findings about metastasis regulation in osteosarcoma should be broadly applicable to many solid tumors. We have data suggesting that Hes1 promotes metastasis by upregulating expression of Metadherin, a cell-surface molecule that can mediate homing of tumor to lung. Our first specific aim will define that regulatory relationship and determine if Metadherin is essential for osteosarcoma metastasis. Since our preliminary work has validated the use of matrigel invasion as a surrogate for metastatic potential in our tumor models, most of our experiments will rely on this in vitro surrogate for metastasis to efficiently evaluate laboratory manipulations of Hes1 and Metadherin. However, we will assess Metadherin knock-down in vivo using two novel xenograft models of metastasis developed in our laboratory. We also have preliminary data showing that epigenetic regulation of the Notch pathway - specifically HDAC inhibition and/or the differential expression of the microRNA34 family - may be the upstream regulators of this novel metastasis promoting signal in osteosarcoma. Specific aim 2 will define whether microRNAs and HDAC inhibitors influence Notch-mediated metastasis, using matrigel invasion as the primary functional assay for invasiveness. Finally, we wish to apply our observations to improve outcomes for patients by identifying those at greatest risk for metastasis. To that end, the third specific aim measures the expression of Notch pathway genes and Metadherin using Q-PCR and immunohistochemistry in a panel of 140 archival tumors from patients treated at U. T. M. D. Anderson and compared expression to outcome, identifying the marker and assay with the greatest prognostic potential. PUBLIC HEALTH RELEVANCE: When successfully completed, these studies will determine how expression of Notch1 and Hes1 are regulated in osteosarcoma, and how this expression relates to expression of Metadherin in controlling invasiveness and metastasis. These mechanisms are likely to be important not only for osteosarcoma, but also for a wide range of solid tumors in which control of metastasis is key to prolonging survival and increasing cure rates. Improved understanding of these basic mechanisms of metastasis should lead to identification of inhibitable components within this new pathway that will serve as the targets for novel therapies to block metastasis.